Release of N‐Acetylaspartylglutamate from Slices of Rat Cerebellum, Striatum, and Spinal Cord, and the Effect of Climbing Fiber Deprivation

Abstract

The release of endogenous N‐acetylaspartylglutamate (NAAG) from slices of rat cerebellum, striatum, and spinal cord upon depolarization with 50 mM K+ was investigated. NAAG in superfusates was prepurified using an ion exchanger, esterified, and then quantified by gas chromatography‐mass spectrometry. Deuterated NAAG was used as internal standard. A depolarization‐induced release of NAAG was found in all three regions. The release was Ca2+ dependent to over 85% in cerebellum and striatum, but only to approximately 70% in spinal cord. In addition, the effect of lesions of the olivocerebellar pathway on the K+‐induced release of NAAG was studied: Treatment of the animals with 3‐acetylpyridine reduced the release of NAAG from cerebellar hemispheres significantly, by about 40% compared with controls. These results suggest that part of the NAAG released from cerebellar slices on depolarization is related to climbing fibers. Implications of these findings concerning possible physiological roles of NAAG in the three CNS regions are discussed.

NAAG Reduces NMDA Receptor Current in CA1 Hippocampal Pyramidal Neurons of Acute Slices and Dissociated Neurons

“…Immunohistochemical studies indicate that NAAG is localized to many putative glutamatergic neuronal systems and to some nonglutamatergic systems (Moffett et al, 1989(Moffett et al, , 1994Frondoza et al, 1990;Slusher et al, 1992;Tsai et al, 1993;Moffett and Namboodiri, 1995;Berger et al, 1995a;Passani et al, 1997). Electrical stimulation of NAAGcontaining axons evokes the release of NAAG in a Ca 2 þ -dependent manner, consistent with its known localization to synaptic vesicles (Williamson and Neale, 1988;Tsai et al, 1988Tsai et al, , 1990Zollinger et al, 1994). NAAG is catabolized to Nacetylaspartate and glutamate by glutamate carboxypeptidase II (GCP II), also known as N-acetyl-alpha-linked acidic dipeptidase (NAALADase; Stauch et al, 1989).…”

Phenotypic characterization of mice heterozygous for a null mutation of glutamate carboxypeptidase II

“…N-acetyl aspartyl glutamate (NAAG) is a neuropeptide, which is concentrated in storage vesicles and released in a calcium-dependent manner upon neuronal depolarization (Williamson and Neale., 1988; Tsai et al, 1988; Zollinger et al, 1994; Renno et al, 1997). NAAG has a highly selective co-localization with classical neurotransmitters such as the cholinergic acid motor neurons, the glutamatergic retinal ganglionic cell projections, the noradrenergic locus coeruleus neurons and the glutamatergic cortical pyramidal neurons, especially in primates (Moffett et al, 1989, 1993, 1994, 1995; Frondoza et al, 1990; Slusher et al, 1992; Tsai et al, 1993; Berger et al, 1995; Passani et al, 1997).…”

Development and application of a liquid chromatography/tandem mass spectrometric assay for measurement of N-acetylaspartate, N-acetylaspartylglutamate and glutamate in brain slice superfusates and tissue extracts

“…The limits of detection achieved using these UV based methods are adequate for measurement of NAAG in brain tissue extracts but these are insufficient for measurement of the analytes in superfusates. Other techniques that have been reported for measurement of NAAG are gas chromatography with mass spectrometry [21,22] and precolumn derivatisation with fluorescence detection [23]. Although the limits of detection obtained using these methods are adequate for measurement of NAAG in superfusates they involve complicated sample preparation steps and the separation time is relatively long.…”